Chemically modified exogenous oligonucleotides (ONs) can bind to specific nucleic acid sequences (i.e., double stranded DNA, single stranded DNA, single stranded RNA), which has enabled their use as fundamental research tools, diagnostic probes and therapeutic agents. For example, modulation of gene expression via the antigene [Duca et al., Nucleic Acids Res. 36:5123 (2008)], antisense [Kurreck, J. Eur. J. Biochem. 270:1628-1644 (2003)] and/or siRNA [Bumcrot et al., Nat Chem Biol 2:711-719 (2006)] strategies are very useful approaches to establish the function of specific genes or transcripts (fundamental research tool), and to develop agents against diseases of genetic origin (therapeutics).
Introduction of chemically modified nucleotides into ONs has been investigated for increasing binding affinity toward double stranded DNA (via Hoogsteen base-pairing), single stranded DNA (via Watson-Crick base-pairing), and single stranded RNA targets (via Watson-Crick base-pairing); b) improving discrimination of mismatched nucleic acid targets to minimize false positives and non-target specific effects in diagnostic and biological applications; and/or c) enhancing stability against degradation by enzymes including nucleases. The use of conformationally restricted nucleotides [Meldgaard et al., J. Chem. Soc., Perkin Trans. 1:3539-3554 (2000); Leumann, J. Bioorg. Med. Chem. 10:841-854 (2002)] such as Locked Nucleic Acid (LNA, also called bridged nucleic, BNA) [Singh et al., Chem. Commun. 455-456 (1998); Koshkin et al., Tetrahedron 54:3607-3630 (1998); Obika et al., Tetrahedron Lett. 39:5401-5404 (1998); Obika et al., Bioorg. Med. Chem. 9:1001-1011 (2001)] or α-L-LNA, [Sørensen et al., J. Am. Chem. Soc. 124:2164-2176 (2002)] have partially addressed these challenges. LNA and α-L-LNA exhibit increased thermal affinity toward complementary single stranded DNA/RNA strands of up to +10° C. per modification, along with markedly improved mismatch discrimination and enzymatic stability relative to unmodified oligodeoxyribonucleotides [Petersen et. al., Trends Biotechnol. 21:74-81 (2003)]. Similarly, introducing LNA or α-L-LNA monomers into triplex forming oligonucleotides may result in markedly improved thermal affinity toward double stranded DNA targets [Torigoe et al., J. Biol. Chem. 276:2354 (2001); Obika et al., Chem. Pharm. Bull. 52:1399 (2004); Sun et al., Biochemistry 43:4160 (2004); Brunet et al., J. Biol. Chem. 280:20076 (2005); Kumar et al., J. Am. Chem. Soc., 128:14 (2006)]. These properties render LNA and α-L-LNA with considerable therapeutic and diagnostic potential [Petersen et. al., Trends Biotechnol. 21:74-81 (2003); Frieden et al., IDrugs 9:706-711 (2006); Jepsen et al., Curr. Opin. Drug Discovery Dev. 7:188-194 (2004); Grünweller et al., Biodrugs 21:235-243 (2007); Stenvang et al., Sem. Cancer Biol. 18:89-102 (2008)].
Substantial efforts have been invested to develop LNA analogs with more desirable biophysical properties, i.e., improved thermal affinity toward single stranded DNA/RNA targets or double stranded DNA targets, improved thermal mismatch discrimination and enhanced enzymatic stability [representative examples: Koizumi et al., Nucleic Acids Res. 31:3267 (2003); Morita et al., Bioorg. Med. Chem. 11:2211-2226 (2003); Sørensen et al., Chem. Commun. 2130-2131 (2003); Fluiter et al., Chem Bio Chem. 1104-1109 (2005); Albaek et al., J. Org. Chem. 71:7731-7740 (2006); Varghese et al., J. Am. Chem. Soc. 128:15173-15187 (2006); Højland et al., Org. Biomol. Chem. 5:2375 (2007); Rahman et al., Angew. Chem., Int. Ed., 46:4306 (2007); Rahman et al., J. Am. Chem. Soc. 130:4886-4896 (2008); Mitsuoka et al., Nucleic Acids Res. 37:1225-1238 (2009); Kumar et al., J. Org. Chem. 1070-1081 (2009); Seth et al., J. Med. Chem. 52:10-13 (2009); Zhou et al., J. Org. Chem. 74:118-134 (2009)]. These studies have primarily focused on using stereoisomers of LNA, modification of the oxymethylene bridge spanning the C2′- and C4′-positions and/or introducing minor-groove oriented substituents into the bridge. Improved enzymatic stability [Morita et al. (2003), Varghese et al. (2006), Rahman et al. (2008), Zhou et al. (2009)], altered biodistribution [Fluiter et al. (2005)], or reduced hepatotoxicity [Seth (2009)], has been reported for some of the analogs, but improvements in hybridization properties relative to LNA were generally not observed. Thus, the significantly increased synthetic complexity of these conformationally restricted nucleotide analogs does not appear to be justified.